An injectable hydrogel with redox activity and electroactivity, and its preparation method and application

Abstract

The present invention discloses an injectable hydrogel with redox activity and electroactivity, as well as its preparation method and application, belonging to the technical field of medical materials. The preparation method includes the following steps: reacting methacrylic anhydride and polysaccharide molecules in a solvent to obtain a double-bond functional polymer; pre-polymerizing dopamine in a solvent first, and then adding MOF for a polymerization reaction to obtain dopamine-modified MOF; reacting dopamine-modified MOF, EDOT and iron salt in a solvent, collecting the precipitate to obtain a nano filler; dispersing the double-bond functional polymer, active polypeptide, photoinitiator and nano filler in a solvent together for photocuring crosslinking to obtain the product. The preparation conditions of the present invention are mild, the gelation is rapid, the method is simple, and the obtained hydrogel has injectability and conductivity, can simulate the catalytic activity of natural enzymes, can achieve the scavenging of reactive oxygen species in the early stage of myocardial ischemia, enhance the electrical conduction in the infarct area, and effectively restore cardiac function.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and particularly relates to an injectable hydrogel with redox activity and electroactivity, and a preparation method and application thereof. Background Art

[0002] Heart failure refers to a clinical syndrome caused by various heart diseases, mainly manifested as a reduced cardiac pumping function. Many cardiovascular diseases can cause weakened diastolic and systolic capabilities of the myocardium, such as hypertension, dilated cardiomyopathy, and valvular heart disease, etc. Heart failure is the end-stage manifestation of various heart diseases. From an epidemiological perspective, the prevalence, incidence, and mortality of heart failure are all very high.

[0003] Currently, there are mainly three clinical therapies for heart failure: drug therapy, left ventricular assist device (LVAD), and heart transplantation. Drug therapy mainly focuses on reducing blood pressure, increasing myocardial contractility (positive inotropic drugs), inhibiting sympathetic nerve activity, and anticoagulation, etc., which can effectively relieve the symptoms of patients, but the long-term medication effect is limited. The left ventricular assist device can improve the hemodynamic situation of patients, but there are risks of various complications, including embolism and infection, etc. Heart transplantation is currently an effective treatment method for patients with end-stage heart failure, but due to the limited number of donors, this treatment method is also restricted.

[0004] The treatment of heart failure with injectable hydrogels is an advanced means based on the concepts of tissue engineering and regenerative medicine. However, most hydrogels lack conductive properties and it is difficult to establish a stable electrical coupling. In addition, for the adverse microenvironment of low pH, high ROS, and inflammatory factor infiltration at the infarcted myocardium, hydrogels lacking the ability to scavenge reactive oxygen species are difficult to improve the survival state of surrounding viable myocardial cells, which is not conducive to inducing myocardial tissue repair and regeneration. Summary of the Invention

[0005] In order to solve the above technical problems, the object of the present invention is to provide an injectable hydrogel with redox activity and electroactivity, and a preparation method and application thereof, so as to solve the problems that when the existing injectable hydrogels are applied to the treatment of heart failure, it is difficult to establish a stable electrical coupling, and it is difficult to improve the survival state of surrounding viable myocardial cells for the adverse microenvironment of low pH, high ROS, and inflammatory factor infiltration at the infarcted myocardium, which is not conducive to inducing myocardial tissue repair and regeneration, etc.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] A preparation method of an injectable hydrogel with redox activity and electroactivity, comprising the following steps:

[0008] (1) React methylacrylic anhydride and polysaccharide molecules in a solvent to obtain a double-bond functional polymer;

[0009] (2) First, dopamine is pre-polymerized in a solvent, and then a metal-organic framework compound is added for polymerization reaction to obtain a dopamine-modified metal-organic framework compound;

[0010] (3) The dopamine-modified metal-organic framework compound, 4-ethylenedioxythiophene, and an iron salt obtained in step (2) are reacted in a solvent, and the precipitate is collected to obtain a nano-filler;

[0011] (4) The double-bond functional polymer, bioactive polypeptide, photoinitiator, and nano-filler obtained in step (3) obtained in step (1) are jointly dispersed in a solvent and subjected to photocuring crosslinking to obtain the product.

[0012] The beneficial effects of the present invention are as follows: The preparation conditions of the present invention are mild, the gelation is rapid, the preparation method is simple, and the obtained hydrogel has excellent injectability and conductivity, can simulate the catalytic activity of natural enzymes, can achieve the scavenging of reactive oxygen species in the early stage of myocardial ischemia, enhance the electrical conduction of the infarcted area, and effectively restore cardiac function.

[0013] Further, in step (1), the polysaccharide molecule is sodium alginate, and the solvent is water; the reaction temperature is 0 - 10 °C, and the time is 20 - 30 h.

[0014] Preferably, in step (1), the reaction temperature is 4 °C, and the time is 24 h.

[0015] Further, in step (1), the concentration of sodium alginate in the reaction solution is 0.5 w / v % - 2 w / v %; the volume ratio of methacrylic anhydride to the solvent is (3 - 10):(80 - 120).

[0016] Preferably, in step (1), the concentration of alginic acid in the reaction solution is 1 w / v %; the volume ratio of methacrylic anhydride to the solvent is 8:100.

[0017] Further, in step (1), the pH of the reaction solution is adjusted to 8 - 9 by a NaOH solution.

[0018] Further, the metal-organic framework compound in step (2) is prepared by the following steps:

[0019] (a) Methyl p-formylbenzoate and pyrrole are refluxed in a solvent to obtain a purple solid product;

[0020] (b) The purple solid product obtained in step (a) and manganese chloride tetrahydrate are refluxed in a solvent, and then after the solution is cooled to room temperature, water is added and stirred, and the precipitate is collected;

[0021] (c) First, dissolve the precipitate obtained in step (b) in an organic solvent, then mix it with an aqueous KOH solution and reflux. Next, remove the organic solvent by rotary evaporation, redissolve it in water, and finally add an acidic solution to precipitate and collect the solid to obtain a purple powder product;

[0022] (d) Mix zirconium oxychloride octahydrate, benzoic acid, and the purple powder product obtained in step (c) in a solvent for reaction, collect the precipitate to obtain the product.

[0023] Further, in step (a), the solvent is propionic acid, and the reflux time is 10 - 15 h;

[0024] In step (b), the solvent is N,N - dimethylformamide, the reflux time is 4 - 8 h, and the stirring time is 30 - 90 min;

[0025] In step (c), the organic solvent is a mixed solvent composed of tetrahydrofuran and methanol, and the mixed reflux time is 10 - 15 h;

[0026] In step (d), the temperature of the mixed reaction is 80 - 100 °C, and the time is 4 - 6 h.

[0027] Preferably, in step (a), the reflux time is 12 h;

[0028] In step (b), the reflux time is 6 h, and the stirring time is 60 min;

[0029] In step (c), the mixed reflux time is 12 h;

[0030] In step (d), the temperature of the mixed reaction is 90 °C, and the time is 5 h.

[0031] Further, in step (a), the mass - volume ratio of methyl p - formylbenzoate, pyrrole, and the solvent is 10 - 20 g : 4 - 8 mL : 150 - 250 mL.

[0032] Preferably, in step (a), the mass - volume ratio of methyl p - formylbenzoate, pyrrole, and the solvent is 14 g : 6 mL : 200 mL.

[0033] Further, in step (b), the mass - volume ratio of the purple solid product, manganese chloride tetrahydrate, and the solvent is 0.4 - 1.0 g : 2.0 - 3.0 g : 50 - 150 mL.

[0034] Preferably, in step (b), the mass - volume ratio of the purple solid product, manganese chloride tetrahydrate, and the solvent is 0.854 g : 2.5 g : 100 mL.

[0035] Further, in step (c), the organic solvent is a mixed solvent composed of tetrahydrofuran and methanol with a volume ratio of (20 - 30):(20 - 30); the mass ratio of the precipitate to KOH is (0.5 - 1.0):(2 - 5).

[0036] Preferably, in step (c), the organic solvent is a mixed solvent composed of tetrahydrofuran and methanol with a volume ratio of 25:25; the mass ratio of the precipitate to KOH is 0.75:2.63.

[0037] Further, in step (d), the mass - volume ratio of zirconium oxychloride octahydrate, benzoic acid, the purple powder product, and the solvent is 30 - 40 mg:250 - 500 mg:10 - 20 mg:10 - 20 mL.

[0038] The beneficial effects of adopting the above - mentioned further technical solutions are as follows: The MOF nanocomposite based on manganese porphyrin highly mimics the coordination structure of natural superoxide dismutase SOD2. As the redox - active center, the manganese - porphyrin metal - organic linker has biomimetic SOD - and CAT - like activities. The manganese porphyrin is assembled orderly as the catalytic unit, and at the same time, the highly positively charged zirconium - oxygen cluster is used as the metal connection point to increase the redox potential of its catalytic center Mn III. The prepared MOF nanocomposite with redox activity can effectively scavenge reactive oxygen species and free radicals.

[0039] Further, in step (2), the solvent is a Tris - HCl solution, the pre - polymerization time is 20 - 40 min, and the polymerization reaction time is 10 - 15 h.

[0040] Preferably, the pre - polymerization time is 30 min, and the polymerization reaction time is 12 h.

[0041] Further, in step (2), the mass - volume ratio of dopamine, the metal - framework compound, and the solvent is 50 - 100 mg:150 - 250 mg:50 - 150 mL.

[0042] Preferably, in step (2), the mass - volume ratio of dopamine, the metal - framework compound, and the solvent is 100 mg:200mg:100 mL.

[0043] Further, in step (3), the solvent is ethanol, the iron salt is ferric chloride hexahydrate, and the reaction condition is stirring in an ice bath for 20 - 30 h.

[0044] Further, in step (3), the mass - volume ratio of the dopamine - modified metal - framework compound, the iron salt, 4 - ethylenedioxythiophene, and the solvent is 250 - 350 mg:3 - 5 g:200 - 250 μL:30 - 50 mL.

[0045] Preferably, in step (3), the mass-volume ratio of the dopamine-modified metal-organic framework compound, iron salt, 4-ethylenedioxythiophene, and solvent is 296.4 mg: 4 g: 228 μL: 40 mL.

[0046] The beneficial effect of adopting the above further technical solution is as follows: In order to solve the problem of poor electrical conductivity of MOF materials, a polydopamine adhesion layer is modified on its surface, and then a conductive polymer is in-situ polymerized. The molecular chain of the conductive polymer serves as a bridge for charge transfer, releases electrons into the electrolyte during the electrochemical process, enhances the Faraday process at the interface, and thus contributes to pseudocapacitance. When this nanofiller is loaded in the hydrogel, it endows the hydrogel with redox activity and electroactivity at the same time.

[0047] Further, the amino acid sequence of the bioactive polypeptide in step (4) is as shown in SEQ ID NO.1:

[0048] CGERGAPGFRGPAGPNGIPGEKGPAGERGAP (SEQ ID NO.1).

[0049] The beneficial effect of adopting the above further technical solution is as follows: In the present invention, the cell adhesion polypeptide is modified on the natural polysaccharide molecular backbone by covalent crosslinking, which enhances the interaction between the hydrogel matrix and cells, improves the affinity between the hydrogel and tissues, and lays a good foundation for the realization of various functions of the hydrogel platform.

[0050] Further, the solvent in step (4) is water;

[0051] The concentration of the double-bond functional polymer in the reaction solution is 1 w / v % - 5 w / v %;

[0052] The concentration of the photoinitiator is 0.1 w / v % - 0.5 w / v %;

[0053] The mass ratio of the bioactive polypeptide to the double-bond functional polymer is 0.1:1 - 1:1;

[0054] The concentration of the nanofiller is 10 - 80 μg / mL;

[0055] The conditions for photocuring crosslinking are: irradiating with ultraviolet light of 350 - 450 nm for 30 - 60 s.

[0056] The beneficial effect of adopting the above further technical solution is as follows: In the present invention, the double bonds are crosslinked into a gel by an addition reaction under ultraviolet light. The gel-forming conditions are mild, the gel formation is rapid, and the preparation method is simple, endowing the hydrogel with excellent rheological properties and injectability.

[0057] An injectable hydrogel with redox activity and electroactivity is prepared by the above preparation method.

[0058] Use of the above injectable hydrogel with redox activity and electroactivity in the preparation of a drug for treating heart failure.

[0059] The present invention has the following beneficial effects:

[0060] (1) By covalently cross-linking and modifying cell adhesion polypeptides on the natural polysaccharide molecular backbone, the present invention enhances the interaction between the hydrogel matrix and cells, improves the affinity between the hydrogel and tissues, and lays a good foundation for the realization of various functions of the hydrogel platform.

[0061] (2) The present invention orderly assembles manganese porphyrin as a catalytic unit, and uses highly positively charged zirconium oxide clusters as metal connection points to increase the redox potential of its catalytic center Mn III. The obtained MOF nanocomposite filler with redox activity can effectively scavenge reactive oxygen species and free radicals.

[0062] (3) By modifying the surface of the MOF material with a polydopamine adhesion layer and then in-situ polymerizing a conductive polymer, the present invention endows the hydrogel with redox activity and electroactivity.

[0063] (4) The present invention has mild gelation conditions, rapid gelation, a simple preparation method, and the prepared hydrogel has excellent rheological properties and injectability.

[0064] (5) The hydrogel prepared by the present invention has good injectability and conductivity, and at the same time has the catalytic activity of mimicking natural enzymes, can scavenge reactive oxygen species in the early stage of myocardial ischemia, enhance the electrical conduction of the infarcted area, and ultimately achieve the purpose of restoring cardiac function. Description of the Drawings

[0065] Figure 1 It is a test chart of the gelation and injectability of the hydrogel prepared in Example 1. Among them, i) and ii) are the detections of gelation, and iii) is the detection of injectability;

[0066] Figure 2 It is a rheological behavior chart of the hydrogel prepared in Example 1. Among them, a is the test result of rheological properties, and b is the test result of self-healing performance;

[0067] Figure 3 It is a test result chart of the conductivity of the hydrogels prepared in Example 1, Example 4-5, and Comparative Example 1;

[0068] Figure 4 It is a microscopic morphology and element distribution chart of the nanocomposite filler PEDOT@PMOF prepared in Example 1. Among them, a is the microscopic morphology chart, and b is the element distribution chart;

[0069] Figure 5Test results graph of the reactive oxygen species scavenging ability of the nano filler PEDOT@PMOF prepared in Example 1. Among them, a is the ultraviolet absorption spectrum of the SOD-like performance of the nano filler PEDOT@PMOF at different concentrations, b is the EPR result of the SOD-like performance of the nano filler PEDOT@PMOF, c is the EPR result of the scavenging efficiency of the nano filler PEDOT@PMOF for hydroxyl radicals, and d is the ultraviolet absorption spectrum of the CAT-like enzyme performance of the nano filler PEDOT@PMOF.

[0070] Figure 6 Graph of the animal treatment ultrasound experiment results of the hydrogel prepared in Example 1;

[0071] Figure 7 Graph of the animal treatment tissue section results of the hydrogel prepared in Example 1;

[0072] Figure 8 Graph of the results that the hydrogel prepared in Example 1 can effectively reduce the susceptibility to arrhythmia. Among them, a-c are the arrhythmia results of the sham operation group, the myocardial infarction model group, and the experimental group under electrical stimulation in sequence, and d is the arrhythmia induction quotient result. Detailed implementation manners

[0073] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0074] Example 1:

[0075] A preparation method of an injectable hydrogel with redox activity and electroactivity, comprising the following steps:

[0076] (1) Synthesis of double bond-functionalized modified alginic acid (AlgMA)

[0077] First, under sterile conditions, prepare 100 mL of a 1 w / v % sodium alginate aqueous solution, then slowly add 8 mL of methacrylic anhydride at 4°C, control the pH value of the solution at 8-9 with 5 mol / L NaOH, after reacting for 24 h, dialyze with water for 3 days, and then freeze-dry for 3 days to obtain AlgMA.

[0078] (2) Synthesis of the bioactive polypeptide T16

[0079] Synthesize the polypeptide T16 with a terminal thiol group by the standard solid-phase synthesis method. The amino acid sequence is SEQ ID NO.1: CGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0080] (3) Synthesis of Metal-organic Framework Compound MOF (PCN222-Mn)

[0081] First, dissolve 1.4 g of methyl 4-formylbenzoate in 200 mL of propionic acid, and slowly add 6 mL of pyrrole dropwise. The mixed solution is refluxed in the dark for 12 h. After the reaction, cool the mixture to room temperature, filter and wash it three times with methanol and tetrahydrofuran, and then filter with suction to obtain a purple solid product A.

[0082] Next, dissolve 0.854 g of product A and 2.5 g of manganese chloride tetrahydrate in 100 mL of DMF. The mixed solution is refluxed in the dark for 6 h. After the reaction, cool the solution to room temperature, add 150 mL of H 2 2O and continuously stir for 1 h. The precipitate B precipitated in the solution is obtained after washing with water and filtering three times.

[0083] Then, dissolve 0.75 g of precipitate B in a mixed solution composed of 25 mL of tetrahydrofuran (THF) and 25 mL of methanol (MeOH). Add 25 mL of an aqueous solution of KOH (2.63 g, 46.95 mmol) to it, and heat the obtained mixture under reflux for 12 h. After the mixture is cooled to room temperature, remove THF and MeOH by rotary evaporation. Add additional water to the system after rotary evaporation, heat the mixture until the solid is completely dissolved, then add 1 mol / L HCl solution to gradually precipitate the solid. Continue to add HCl solution until no new solid precipitates. Filter and collect the precipitate, wash it with water and dry it under vacuum to obtain a purple powder product C.

[0084] Finally, dissolve zirconium oxychloride octahydrate (30 mg), benzoic acid (0.28 g) and product C (10.98 mg) in 14 mL of N,N-dimethylformamide (DMF), stir at 90 °C for 5 h. After the reaction, cool to room temperature, collect by centrifugation, and wash the nanoparticles three times with DMF and deionized water respectively to obtain PCN22-Mn.

[0085] (4) Preparation of Dopamine-modified PCN222-Mn

[0086] First, dissolve 100 mg of dopamine in 100 mL of Tris-HCl solution with pH = 8.5, carry out pre-polymerization for 30 min, then add 200 mg of MOF to the pre-polymerization solution, polymerize for 12 h, and finally wash it three times with ethanol to obtain dopamine-modified PCN222-Mn, denoted as PMOF.

[0087] (5) Preparation of Nanofiller PEDOT@PMOF

[0088] First, PMOF (296.4 mg) and EDOT (228 μL) were dispersed in ethanol (30 mL) under strong stirring, and then an ethanol solution of ferric chloride hexahydrate (4 g, 10 mL) was added dropwise. The mixture was stirred for 24 h in an ice bath. Finally, after washing with deionized water and centrifuging, the nano-filler PEDOT@PMOF was obtained.

[0089] (6) Preparation of hydrogel

[0090] AlgMA (50 mg), bioactive polypeptide T16 (5 mg), photoinitiator LAP (5 mg), and PEDOT@PMOF nano-filler (20 μg) were uniformly dispersed in 1 mL of water, and then irradiated with ultraviolet light at 405 nm for 45 s to crosslink into a gel, obtaining an injectable hydrogel with redox activity and electroactivity.

[0091] Example 2:

[0092] A preparation method of an injectable hydrogel with redox activity and electroactivity, comprising the following steps:

[0093] (1) Synthesis of double bond-functionalized modified alginic acid (AlgMA)

[0094] First, under sterile conditions, a 100 mL 1 w / v % aqueous sodium alginate solution was prepared, and then 5 mL of methacrylic anhydride was slowly added at 4 °C. The pH value of the solution was controlled at 8 - 9 with 5 mol / L NaOH. After reacting for 24 h, it was dialyzed with water for 3 days, and then freeze-dried for 3 days to obtain AlgMA.

[0095] (2) Synthesis of bioactive polypeptide T16

[0096] The polypeptide T16 with a terminal mercapto group was synthesized by the standard solid-phase synthesis method. The amino acid sequence is SEQ ID NO.1: CGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0097] (3) Synthesis of metal-organic framework compound MOF (PCN222-Mn)

[0098] First, 1.4 g of methyl 4-formylbenzoate was dissolved in 200 mL of propionic acid, and 8 mL of pyrrole was added dropwise. The mixed solution was refluxed in the dark for 12 h; after the reaction ended, the mixture was cooled to room temperature and filtered and washed three times with methanol and tetrahydrofuran, and then suction-filtered to obtain a purple solid product A.

[0099] Then, 0.427 g of product A and 2.0 g of manganese chloride tetrahydrate were dissolved in 100 mL of DMF, and the mixed solution was refluxed in the dark for 6 h; after the reaction ended, the solution was cooled to room temperature, and 150 mL of H2 O and continuously stir for 1 h; the precipitate B precipitated in the solution is obtained by washing with water and filtering three times.

[0100] Then dissolve 0.75 g of precipitate B in a mixed solution composed of 25 mL of tetrahydrofuran (THF) and 25 mL of methanol (MeOH). Add 25 mL of an aqueous solution of KOH (3.95 g) to it, and heat the resulting mixture under reflux for 12 h. After the mixture is cooled to room temperature, remove THF and MeOH by rotary evaporation; add additional water to the system after rotary evaporation, heat the mixture until the solid completely dissolves, then add 1 mol / L HCl solution to gradually precipitate the solid, continue to add HCl solution until no new solid precipitates, filter and collect the precipitate, wash it with water and dry it under vacuum to obtain the purple powder product C.

[0101] Finally, dissolve zirconium oxychloride octahydrate (30 mg), benzoic acid (0.4 g) and product C (10.98 mg) in 14 mL of N,N-dimethylformamide (DMF), stir at 90 °C for 5 h, cool to room temperature after the reaction, collect by centrifugation, and wash the nanoparticles three times each with DMF and deionized water to obtain PCN22-Mn.

[0102] (4) Preparation of dopamine-modified PCN222-Mn

[0103] First, dissolve 75 mg of dopamine in 100 mL of Tris-HCl solution with pH = 8.5, carry out pre-polymerization for 30 min, then add 200 mg of MOF to the pre-polymerization solution, polymerize for 12 h, and finally wash three times with ethanol to obtain dopamine-modified PCN222-Mn, denoted as PMOF.

[0104] (5) Preparation of nanofiller PEDOT@PMOF

[0105] First, disperse PMOF (296.4 mg) and 4-ethylenedioxythiophene (EDOT, 228 μL) in ethanol (30 mL) under strong stirring, then dropwise add an ethanol solution of ferric chloride hexahydrate (4 g, 10 mL), stir under ice bath for 24 h, and finally obtain the nanofiller PEDOT@PMOF after washing with deionized water and centrifugation.

[0106] (6) Preparation of hydrogel

[0107] AlgMA (50 mg), active polypeptide T16 (5 mg), photoinitiator LAP (2.5 mg) and PEDOT@PMOF nanofiller (20 μg) were uniformly dispersed in 1 mL of water, and then irradiated with ultraviolet light at 405 nm for 30 s to crosslink into a gel, obtaining an injectable hydrogel with redox activity and electroactivity.

[0108] Example 3:

[0109] A preparation method of an injectable hydrogel with redox activity and electroactivity, comprising the following steps:

[0110] (1) Synthesis of double bond-functionalized modified alginic acid (AlgMA)

[0111] First, under sterile conditions, prepare 100 mL of a 1 w / v % sodium alginate aqueous solution, then slowly add 3 mL of methacrylic anhydride at 4 °C, control the pH value of the solution at 8 - 9 with 5 mol / L NaOH, after reacting for 24 h, dialyze with water for 3 days, and then freeze-dry for 3 days to obtain AlgMA.

[0112] (2) Synthesis of active polypeptide T16

[0113] The polypeptide T16 with a terminal thiol group was synthesized by the standard solid-phase synthesis method. The amino acid sequence is SEQ ID NO.1: CGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0114] (3) Synthesis of metal-organic framework compound MOF (PCN222-Mn)

[0115] First, dissolve 1.4 g of methyl 4-formylbenzoate in 200 mL of propionic acid, and slowly add 4 mL of pyrrole dropwise. The mixed solution was refluxed in the dark for 12 h; after the reaction, the mixture was cooled to room temperature, filtered and washed three times with methanol and tetrahydrofuran, and the purple solid product A was obtained by suction filtration.

[0116] Then, dissolve 0.854 g of product A and 3.0 g of manganese chloride tetrahydrate in 100 mL of DMF, and reflux the mixed solution in the dark for 6 h; after the reaction, the solution was cooled to room temperature, add 150 mL of H 2 O and stir continuously for 1 h; the precipitate B precipitated in the solution was obtained by washing with water and filtering three times.

[0117] Then, 0.5 g of precipitate B was dissolved in a mixed solution composed of 25 mL of tetrahydrofuran (THF) and 25 mL of methanol (MeOH). 25 mL of an aqueous solution of KOH (2.63 g, 46.95 mmol) was added thereto. The resulting mixture was heated under reflux for 12 h. After the mixture was cooled to room temperature, THF and MeOH were removed by rotary evaporation. Additional water was added to the system after rotary evaporation, and the mixture was heated until the solid completely dissolved. Then, 1 mol / L HCl solution was added to gradually precipitate the solid. HCl solution was continuously added until no new solid was precipitated. The precipitate was collected by filtration, washed with water, and dried in vacuo to obtain the purple powder product C.

[0118] Finally, zirconium oxychloride octahydrate (50 mg), benzoic acid (0.5 g), and product C (18.3 mg) were dissolved in 14 mL of N,N-dimethylformamide (DMF). The mixture was stirred at 90 °C for 5 h. After the reaction, it was cooled to room temperature, centrifuged for collection, and the nanoparticles were washed three times with DMF and deionized water respectively to obtain PCN22-Mn.

[0119] (4)Preparation of dopamine-modified PCN222-Mn

[0120] First, 50 mg of dopamine was dissolved in 100 mL of Tris-HCl solution with pH = 8.5 for 30 min of prepolymerization. Then, 200 mg of MOF was added to the prepolymerization solution and polymerized for 12 h. Finally, it was washed three times with ethanol to obtain dopamine-modified PCN222-Mn, denoted as PMOF.

[0121] (5)Preparation of nanofiller PEDOT@PMOF

[0122] First, PMOF (296.4 mg) and EDOT (228 μL) were dispersed in ethanol (30 mL) under strong stirring. Then, an aqueous solution of ferric chloride hexahydrate in ethanol (4 g, 10 mL) was added dropwise, and the mixture was stirred for 24 h in an ice bath. Finally, it was washed with deionized water and centrifuged to obtain the nanofiller PEDOT@PMOF.

[0123] (6)Preparation of hydrogel

[0124] AlgMA (50 mg), bioactive polypeptide T16 (10 mg), photoinitiator LAP (5 mg), and PEDOT@PMOF nanofiller (20 μg) were uniformly dispersed in 1 mL of water, and then irradiated with ultraviolet light at 405 nm for 60 s to crosslink into a gel, obtaining an injectable hydrogel with redox activity and electroactivity.

[0125] Example 4:

[0126] A preparation method of an injectable hydrogel with redox activity and electroactivity, comprising the following steps:

[0127] The preparation method is the same as that of Example 1, except that the mass of the PEDOT@PMOF nanofiller in step (6) is changed to 10 μg.

[0128] Example 5:

[0129] A preparation method of an injectable hydrogel with redox activity and electroactivity, comprising the following steps:

[0130] The preparation method is the same as that of Example 1, except that the mass of the PEDOT@PMOF nanofiller in step (6) is changed to 40 μg.

[0131] Comparative Example 1:

[0132] A preparation method of an injectable hydrogel, comprising the following steps:

[0133] (1) Synthesis of double bond-functionalized alginic acid (AlgMA)

[0134] First, under sterile conditions, prepare a 100 mL 1 w / v% sodium alginate aqueous solution, then slowly add 8 mL of methacrylic anhydride at 4°C, control the pH value of the solution at 8 - 9 with 5 mol / L NaOH, after reacting for 24 h, perform water dialysis for 3 days, and then freeze-dry for 3 days to obtain AlgMA.

[0135] (2) Synthesis of the bioactive polypeptide T16

[0136] Synthesize the polypeptide T16 with a terminal thiol group using the standard solid-phase synthesis method. The amino acid sequence is SEQ ID NO.1: CGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0137] (3) Preparation of the hydrogel

[0138] Disperse AlgMA (50 mg), the bioactive polypeptide T16 (5 mg), and the photoinitiator LAP (5 mg) uniformly in 1 mL of water, and then irradiate with ultraviolet light at 405 nm for 45 s to crosslink into a gel to obtain the injectable hydrogel TAlg.

[0139] Comparative Example 2:

[0140] A preparation method of an injectable hydrogel, comprising the following steps:

[0141] (1) Synthesis of double bond-functionalized alginic acid (AlgMA)

[0142] First, under aseptic conditions, prepare 100 mL of 1 w / v % sodium alginate aqueous solution. Then, at 4 °C, slowly add 8 mL of methacrylic anhydride, and use 5 mol / L NaOH to control the pH value of the solution at 8 - 9. After reacting for 24 h, dialyze with water for 3 days, and then freeze-dry for 3 days to obtain AlgMA.

[0143] (2)Synthesis of bioactive polypeptide T16

[0144] Synthesize the polypeptide T16 with a terminal thiol group using the standard solid-phase synthesis method. The amino acid sequence is SEQ ID NO.1: CGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0145] (3)Synthesis of metal-organic framework compound MOF (PCN222-Mn)

[0146] First, dissolve 1.4 g of methyl 4-formylbenzoate in 200 mL of propionic acid, and slowly add 6 mL of pyrrole dropwise. The mixed solution is refluxed in the dark for 12 h. After the reaction is completed, cool the mixture to room temperature, filter and wash it three times with methanol and tetrahydrofuran, and then filter with suction to obtain a purple solid product A.

[0147] Next, dissolve 0.854 g of product A and 2.5 g of manganese chloride tetrahydrate in 100 mL of DMF, and reflux the mixed solution in the dark for 6 h. After the reaction is completed, cool the solution to room temperature, add 150 mL of H 2 2O and continuously stir for 1 h. The precipitate B precipitated in the solution is obtained by washing with water and filtering three times.

[0148] Then, dissolve 0.75 g of precipitate B in a mixed solution composed of 25 mL of tetrahydrofuran (THF) and 25 mL of methanol (MeOH). Add 25 mL of KOH (2.63 g, 46.95 mmol) aqueous solution to it, and heat the resulting mixture to reflux for 12 h. After the mixture is cooled to room temperature, remove THF and MeOH by rotary evaporation. Add additional water to the system after rotary evaporation, heat the mixture until the solid completely dissolves, then add 1 mol / L HCl solution to gradually precipitate the solid. Continue to add HCl solution until no new solid precipitates. Filter and collect the precipitate, wash it with water and dry it under vacuum to obtain a purple powder product C.

[0149] Finally, dissolve zirconium oxychloride octahydrate (30 mg), benzoic acid (0.28 g), and product C (10.98 mg) in 14 mL of N,N-dimethylformamide (DMF), stir at 90 °C for 5 h. After the reaction, cool to room temperature, collect by centrifugation, and wash the nanoparticles three times each with DMF and deionized water to obtain PCN22-Mn.

[0150] Preparation of Dopamine-Modified PCN222-Mn

[0151] First, 100 mg of dopamine was dissolved in 100 mL of Tris-HCl solution with pH = 8.5 for 30 min of prepolymerization. Then, 200 mg of MOF was added to the prepolymerization solution and polymerized for 12 h. Finally, it was washed three times with ethanol to obtain dopamine-modified PCN222-Mn, denoted as PMOF.

[0152] (5)Preparation of Hydrogel

[0153] AlgMA (50 mg), bioactive polypeptide T16 (5 mg), photoinitiator LAP (5 mg) and PMOF (20 μg) were uniformly dispersed in 1 mL of water, and then irradiated with ultraviolet light at 405 nm for 45 s to crosslink into a gel, obtaining an injectable hydrogel with redox activity and electroactivity.

[0154] Test Examples:

[0155] I. Gelation and Injectability

[0156] The gelation performance of the hydrogel prepared in Example 1 was detected by needle injection.

[0157] The experimental results are as Figure 1 shown. The results show that the hydrogel prepared in Example 1 of the present invention can be injected out from a 27G needle, proving that the hydrogel prepared in the present invention has good injectability.

[0158] II. Mechanical Properties and Conductive Properties

[0159] The rheological properties of the hydrogel prepared in Example 2 of the present invention were tested by an MCR302 rheometer. A double concentric cylinder geometry with a gap of 1 mm was used for steady-state shear flow at 37°C. The oscillation frequency of the strain sweep was 1 Hz, and the strain was 0.1 - 1000%. In the self-healing experiment, an alternating step strain sweep experiment (large strain: 1000%, small strain: 1%) was adopted.

[0160] The experimental results are as Figure 2 shown. The results indicate that the hydrogel prepared in Example 2 of the present invention has excellent rheological properties and self-healing ability.

[0161] When conductive nanofillers (such as metal nanoparticles, carbon nanotubes, graphene, and MXene) are uniformly dispersed in the hydrogel matrix, a connected electron path can be formed, thereby enhancing the overall conductivity of the hydrogel. The resistivity of the hydrogels prepared in Example 1, Examples 4 - 5, and Comparative Example 1 was measured by the four-probe method.

[0162] The experimental results are asFigure 3 As shown, the results indicate that, compared with Comparative Example 1, the conductivity of the TAlg / PEDOT@PMOF hydrogel prepared in the embodiment of the present invention is similar to that of healthy myocardium (~10 -4 S cm -1 ). This hydrogel can be electrophysiologically coupled with the myocardium, improve cardiac function, and avoid inducing arrhythmia. Among them, the conductivity of the hydrogel prepared in Example 5 of the present invention is 7.78 ± 0.06×10 -4 S cm -1 .

[0163] III. Detection of the Morphology and Catalytic Activity of Nanofillers

[0164] The nanofiller PEDOT@PMOF prepared in Example 1 was characterized by microscopic morphology, and the elemental composition and distribution of PEDOT@PMOF were analyzed by the high-angle annular dark field (HAADF) mode of STEM.

[0165] The results are as Figure 4 shown. The uniform distribution of elements such as Mn and S in the structure of the nanofiller PEDOT@PMOF can be clearly observed, which proves the existence of Mn element in the MOF structure and the successful polymerization of PEDOT in the pores and on the surface of the MOF.

[0166] In vitro, the SOD-like performance of the nanofiller PEDOT@PMOF was tested in a reaction system of xanthine and xanthine oxidase. Different nanomaterials (20 μg mL -1 ) were respectively mixed with xanthine (0.15 mmol / L) and xanthine oxidase (0.02 U mL -1 ) in a Tris-HCl buffer solution with a pH value of 7.0. After co-incubation at room temperature for 5 min, 0.05 mmol / L of NBT was added to each mixed solution. The superoxide generated by the reaction reduces the color reagent NBT to blue formazan, which has a strong absorption at 560 nm in ultraviolet light. As shown in Figure a in Figure 5 , as the concentration of PEDOT@PMOF increases, the absorption peak intensity weakens and the content of superoxide anions decreases, indicating that the stronger the SOD-like activity, which proves its dose-dependent SOD-like activity. At the same time, the EPR technique was used to investigate the scavenging efficiency of the nanofiller on superoxide radicals. As shown in Figure 5As shown in Figure b, DMPO, a common radical scavenger, can capture short-lived superoxide radicals in methanol solvent. In the system without adding nanofillers, the peak signal of superoxide radicals is strong, showing a typical six-peak structure of four large peaks and two small peaks. After adding three kinds of nanofillers respectively, the peak intensity decreased significantly, and with the extension of the test time, the superoxide radical signal gradually weakened. The unstable superoxide anion combines with hydrogen ions and is converted into hydroxyl radicals. In vitro, hydroxyl radicals are generated based on the Fenton reaction system and then react with salicylic acid to produce a chromogenic group. As Figure 5 As shown in Figure a, with the addition of PEDOT@PMOF and the increase of its concentration, the maximum absorption wavelength intensity at 510 nm gradually decreases. As Figure 5 The EPR results in Figure c show that after 6 min of microwave irradiation, four peaks characteristic of hydroxyl radicals are detected, and the peak intensities from left to right are 1:2:2:1. After adding MOF, PMOF, and PEDOT@PMOF respectively, the radical signal weakens in turn, and the signal basically disappears in the spectrum of the PEDOT@PMOF group at the 12th min. Under pathological conditions, the process of converting intracellular oxygen molecules into water molecules is blocked, and superoxide anions combine with hydrogen ions and are catalyzed by superoxide dismutase to generate hydrogen peroxide. In vitro, based on the redox reaction between titanium sulfate and hydrogen peroxide and the optical properties of its product peroxide-titanium complex, the CAT-like enzyme performance of a series of prepared nanomaterials was detected. As Figure 5 As shown in Figure d, the absorbance value at about 405 nm decreases with the increase of the concentration of PEDOT@PMOF.

[0167] IV. Detection of in vivo effectiveness of hydrogels

[0168] To study the effect of injectable hydrogels with redox activity and electroactivity on repairing heart damage in vivo, a rat myocardial infarction disease model was established by permanently ligating the left anterior descending branch of the heart, and echocardiography was performed on the successfully modeled myocardial infarction rats on the 7th, 14th, and 28th days.

[0169] The hydrogels prepared by Example 1, Comparative Example 1, and Comparative Example 2 were used to implant and treat the model, and 30 μL of hydrogel was injected into two parts, the infarct center area and the infarct edge, with a 27G needle.

[0170] The detection results of M-mode echocardiography are as Figure 6 shown. Compared with the control group, the systolic-diastolic movement of the ventricular wall in the hydrogel group 3 was significantly improved; the Masson staining results on the 28th day are as Figure 7As shown, compared with the control group, the ventricular wall thickness of the heart tissue in the hydrogel group prepared in Example 1 of the present invention is the highest, and the scar area is the smallest, indicating that the hydrogel prepared in Example 1 effectively reduces the scar area at the myocardial infarction site, proving its good heart repair function.

[0171] V. Detection of the susceptibility of hydrogels to reduce arrhythmia in vivo

[0172] Four weeks after treatment by injecting the hydrogel, a standard clinical programmed electrical stimulation (PES) protocol was used to evaluate the inducibility of arrhythmia. The PES study was carried out by an independent programmable stimulator, and the arrhythmia induction rate was quantified as an induction quotient according to a recognized scoring system. The higher the score, the more likely arrhythmia is to occur.

[0173] The experimental results are as Figure 8 shown. Under programmed electrical stimulation, the arrhythmia induction quotient of the rats treated with the hydrogel prepared in Example 1 of the present invention is the lowest, approaching that of the sham operation group. The experimental results show that the hydrogel in the third group has good electrical coupling performance and can promote the electrical conduction between myocardial tissues.

[0174] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an injectable hydrogel having redox activity and electroactivity, characterized in that: The following steps are involved: (1) Methacrylic anhydride and polysaccharide molecules are reacted in a solvent to prepare a double-bond functional polymer; (2) Dopamine is first prepolymerized in a solvent, and then a metal framework compound is added to carry out a polymerization reaction to obtain a dopamine-modified metal framework compound; (3) reacting the dopamine-modified metal framework compound obtained in step (2), 4-ethylenedioxythiophene and iron salt in a solvent, collecting the precipitate, and preparing a nanofiller; (4) dispersing the double-bond functional polymer obtained in step (1), the active polypeptide, the photoinitiator and the nanofiller obtained in step (3) in a solvent to obtain a reaction solution, and performing photocuring and cross-linking to obtain; The metal framework compound in step (2) is prepared by the following steps: (a) reflux methyl p-formylbenzoate and pyrrole in a solvent to obtain a purple solid product; (b) refluxing the purple solid product obtained in step (a) and manganese chloride tetrahydrate in a solvent, then adding water and stirring after the solution is cooled to room temperature, and collecting the precipitate; (c) first dissolving the precipitate obtained in step (b) in an organic solvent, then mixing with a KOH aqueous solution and refluxing, then removing the organic solvent by rotary evaporation, adding water for redissolution, and finally adding an acidic solution to precipitate and collect a solid to obtain a purple powder product; (d) zirconium oxychloride octahydrate, benzoic acid and the purple powder product obtained in step (c) are mixed in a solvent for reaction, and the precipitate is collected to obtain; The amino acid sequence of the active polypeptide in step (4) is shown in SEQ ID NO.

1.

2. The method for preparing the injectable hydrogel having redox activity and electroactivity according to claim 1, characterized in that: In the step (1), the polysaccharide molecule is sodium alginate, the solvent is water, the reaction temperature is 0-10°C, and the reaction time is 20-30 h.

3. The method for preparing the injectable hydrogel having redox activity and electroactivity according to claim 1, characterized in that: In step (a), the solvent is propionic acid and the reflux time is 10-15 h; In step (b), the solvent is N,N-dimethylformamide, the reflux time is 4-8 h, and the stirring time is 30-90 min; In step (c), the organic solvent is a mixed solvent consisting of tetrahydrofuran and methanol, and the mixing and reflux time is 10-15 hours; The temperature of the mixed reaction in step (d) is 80-100° C. and the time is 4-6 h.

4. The method for preparing the injectable hydrogel having redox activity and electroactivity according to claim 1, characterized in that: In the step (2), the solvent is a Tris-HCl solution, the prepolymerization time is 20-40 min, and the polymerization reaction time is 10-15 h.

5. The method for preparing the injectable hydrogel having redox activity and electroactivity according to claim 1, characterized in that: In step (3), the solvent is ethanol, the iron salt is ferric chloride hexahydrate, and the reaction conditions are stirring in an ice bath for 20-30 hours.

6. The method for preparing the injectable hydrogel having redox activity and electroactivity according to claim 1, characterized in that: In step (4), the solvent is water; The concentration of the double bond functional polymer in the reaction solution is 1 w / v %-5 w / v %; The concentration of the photoinitiator is 0.1 w / v %-0.5 w / v %; The mass ratio of active polypeptide to double-bond functional polymer is 0.1:1-1:1; The concentration of nanofillers was 10-80 μg / mL; The conditions for photocuring crosslinking are: irradiation with 350-450 nm ultraviolet light for 30-60 s.

7. An injectable hydrogel having redox activity and electroactivity, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the injectable hydrogel having redox activity and electroactivity according to claim 7 in the preparation of a medicament for treating heart failure.

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